Summary The immune-suppressive microenvironment generated by tumors is a key barrier to immune clearance and thereby, enables cancer to manifest. We propose to overcome this barrier using a plant virus-like nanopar- ticle (VLP) platform technology for in situ vaccination, to eliminate local immunosuppression and generate ef- fective local and systemic anti-tumor immunity. We recently demonstrated that plant-produced, engineered VLP-based nanotechnologies stimulate a potent anti-tumor immune response in mouse models of metastatic melanoma, ovarian cancer, and breast cancer. Data indicate that the effect is systemic and durable, resulting in immune-memory and protection from recurrence. Preliminary studies in companion dogs with metastatic melanoma indicate that the potent anti-tumor efficacy can be replicated in the canine model, which has high relevance to human melanoma. In situ vaccination provides a personalized treatment approach by relieving the patient's tumor-mediated immunosuppression and potentiating anti-tumor immunity against antigens ex- pressed by their own tumor. The proposed nanoengineering approach using plant VLPs would improve the standard of care in several ways. First, in situ vaccination will increase the frequency of antigen-specific T cells, leading to long-lasting immunologic memory; this is in contrast to checkpoint inhibitors, which are not an- tigen specific, activate all antigen-experienced T cells and result in off-target immune toxicities. Second, FDA- approved in situ vaccination using T-VEC demonstrates the approach but is limited because of the use of at- tenuated herpes virus, which can be infectious, has poor stability and an extraordinary price per dose. While our supporting data indicate potent efficacy in various tumor models, the underlying immunology is quite unique and not yet completely understood. Therefore, the essence of this proposal is to decipher the engineering design space of the potent nanotechnology and to delineated the underlying mecha- nism of action. Therefore, this proposal sets out to fulfill the following aims: 1) To decipher the VLP's molecu- lar features triggering the potent efficacy. 2) To delineate the underlying mechanisms of immune activation primed by the VLPs. 3) To gain further insights into the mechanism of action and efficacy through study of companion dogs with oral melanoma. A multi-PI partnership and collaborating investigators will contribute to the success of the program. Together, data will provide detailed mechanisms of anti-tumor immune activation by engineered VLP nanotechnologies and will inform nanoengineering to further improve the approach and begin the effort to test it in human patients.